Antimicrobial wound dressing

ABSTRACT

An antimicrobial wound dressing and method of wound treatment, the wound dressing having a layer of a collagen dressing material impregnated with lyophilized, stabilized chlorine-containing compounds which generate on activation chlorine dioxide, like a mixture of sodium chlorate and sodium chlorite, and an adjacent layer secured thereto containing a dry, activating amount of an acidic compound, such as citric acid, whereby moisture from the wound activates the dry chlorine moiety to treat the wound.

This is a divisional of copending application Ser. No. 07/439,472 filedon Nov. 21, 1989, pending.

BACKGROUND OF THE INVENTION

Wound infection followed by a breakdown of surgical or traumatic woundsstill remains in the forefront of wound care, despite several availablemeasures to prevent this complication of would healing, oftenthreatening the life of the patient.

It has been documented that if the bacterial counts in the wound exceed10⁵ organisms/1 gram of the wound tissue, the infection is a seriousproblem that without proper medical attention results in systemicsepsis. Microorganism infect wounds originating either from within thehuman body, which is a known reservoir for pathogenic organisms, or fromenvironmental origin (military wounds). The most common microorganismsare S. aureus, St. epidermis, beta haemolytic Streptococci, E. coli,Klebsiella and Pseudomonas species and among the anaerobic bacteria, theClostridium welchii or tartium, which are the cause of gas gangrene,mainly in deep traumatic wounds.

It is quite understandable that through the history of wound care,several methods and drugs were tested and used to prevent or reduce therisk of infected wounds. Generally, it can be said that many of theantimicrobial treatments adversely affect the wound and the treatmentbecomes toxic not only to microorganisms but also the cell-wound tissue.Another limitation is that many treatments affect only the surface ofthe wound while some common bacteria (Pseudomonas) quickly merge intothe repair tissue, forming multiple foci with puss. This is a situationthat occurs with very popular drugs based on silver, iodine and cerium,which link actively with proteins of the wound tissue without diffusingdeep enough through the wound to reach the microorganism.

Antibiotics, both systemically or topically administered, represented amilestone in the treatment of infected wounds. However, antibiotics, perse, may represent another "toxic" burden to a patient with multipleinjuries, deep burns over a large body area, or where the liver functionis stressed by the wound toxins. Only topical administration ofantibiotics may result in formation of bacterial strain resistant toadditional treatment with antibiotics.

SUMMARY OF THE INVENTION

The present invention relates to an antimicrobial wound dressing and tothe method of manufacturing and to the method of treating a wound by theuse of the wound dressing.

The antimicrobial wound dressing of the invention comprises a multiplelayer, preferably a two-layer, dressing with one layer retainingmaterial impregnated with an effective, but antimicrobial amount of afreeze-dried, stabilized chlorine-containing compound which onactivation generates chlorine dioxide in situ as an active ingredient,either along or with other antimicrobial materials, while anotheradjoining but separate layer comprises a retaining material impregnatedwith a pharmaceutically-accepted, activating compound, such as a dry,acidic compound in an activating amount for the chlorine compound. Theacidic compound when placed in contact with the freeze dried orlyophilized chlorine-containing compound and in the presence of moisturefrom the wound or externally applied provides for the in situ generationof strong oxidizing chlorine dioxide and hypochlorite for the treatmentof the wound. Generally, the adjacent layer surfaces are securedtogether, such as by the employment of a water permeable, adhesive, andoptionally and preferably, an outer barrier, water and gas impervious,covering layer is employed, such as low vapor permeable material tolimit the drying of the wound to be treated to which the wound dressingis to be applied.

The method of manufacturing the novel wound dressing of the inventioncomprises impregnating a first retaining, sheet type material, such assponge type material, with an antimicrobial amount of a stabilizedchlorine compound containing solution and thereafter freeze drying orlyophilizing the impregnated sheet material to provide dry, chlorinemoieties, impregnated sheet material; and impregnating a secondretaining sheet material which may be the same or different materialthan the first material with an effective, activating amount of anactivator acidic compound, which is pharmaceutically acceptable, such asa low molecular weight, non-toxic, organic acid, such as boric acid,citric acid or ascorbic acid, typically by impregnating the retainingmaterial with a solution of the acid and thereafter, freeze drying orlyophilizing to obtain a retaining material impregnated with the acidicactivator compound. The method optionally includes securing the firstchlorine based retaining material as a separate layer to the second dryretaining material impregnated with the dry, acidic, activator compound,such as by adhesively securing the layers together with a waterpermeable layer, and also optionally, providing a cover barrier sheetover the one top surface of the dry, acidic, activated compound whichforms the upper layer of the wound dressing to prevent the drying out ofthe wound, and optionally, but preferably, sterilizing the multi-layeredwound dressing so prepared.

In use, the wound dressing is placed over the wound by the use ofadhesive tape or the adhesive tape may be a portion of the top coveringsurface with the dry, stabilized, chlorine-containing compounds and theimpregnated retaining material placed adjacent the wound. The moisturein the wound provides together with the dry, acidic, activator compoundand the dry, stable, active components, the conditions for thegeneration of a strong oxidizing and antimicrobial effect of chlorinedioxide and hypochlorite from the lower layer of the retaining materialso as directly to treat in situ and disinfect the wound.

The retaining materials employed in the upper layer of the wounddressing may be the same or different, and typically, such materialcomprises a soft, flexible, porous type material suitable for use incontact with the wound and used as a wound dressing to include, but notbe limited to: natural materials, such as collagen sponge; open cellfoam materials, such as natural and artificial rubber and urethane typefoam materials; membrane materials; woven or non-woven fabrics made ofnatural or synthetic materials; or any other material or substance whichcan retain the dry, impregnated, stabilized chlorine-containingcompounds and the dry, impregnated acidic activator compounds asdesired. A separate layer of the dry active-oxidizing, moiety-producingcompounds and the dry activator compound typically are secured togetherby an adhesive layer, but does not prevent the moisture activation ofthe dry, microbiologically active components from its function in thewound dressing.

The active ingredient in the lower layer is the freeze dried orlyophilized, chlorine-containing compounds, such as sodium chlorites,chlorous acid, chlorates, etc., which generate hypochlorite or chlorinedioxide, impregnated into the retaining material. However, other typesof disinfecting drugs or antibiotics and other materials which do notinterfere with the dry, lyophilized, chlorine-containing, stabilizedcompounds may be used in combination with the dry, lyophilized activecomponents as desired. The acidic activator compound may comprise anyactivator which provides for a weak acidic pH in the presence of wateror moisture so as to activate and convert the dry, lyophilized chlorinecompounds into a strong, oxidizing moiety or chlorine dioxide orhypochlorite through the use of the activator compound. Generally, theactivator compound should be topically, that is, dermatologically andpharmaceutically acceptable, for use on and about wound dressing.Particular compounds which can be used include: citric acid; ascorbicand tartaric acid; boric acid; and similar types of weak acid or otherpharmaceutically-acceptable acid salts. Chemically weak organic acidsare preferable, as these only destabilize the originally stablechlorine-containing compounds, thus inducing slow chemical reactionresulting in sustained, not explosive, formation of strong, oxidizingcompounds such as CLO₂ or CLO.

The dimensions of the sheet material employed in the coverings as wellas the concentrations of the various active and activator ingredientsmay of course vary as desired; however, for example, where a collagensheet is employed as the dry chlorine compound retaining material,typically, such material would have a range of about 0.4 mm to 1.5 mmthick wherein the concentration of the stabilized chlorine dioxide wouldrange from about 0.5 to about 1.5 weight percent of the retainingmaterial. The other retaining layer may be of similar thickness andgenerally may comprise from about 2% to 7% by weight of an active acidsolution prior to freeze drying.

The wound dressing may be packaged in a separate package or theactivated layer containing the dry, stable chlorine components may besealed using a removable, impermeable sealing strip which is removedprior to application to the wound or which may have perforations thereinand be applied directly to the wound so the oxidizing chlorine and otherformed compounds will pass through the perforations and into contactwith the wound. Generally, the wound dressing should be sterilized byany known sterilization method technique, such as ethylene oxide orgamma radiation or other such techniques. Usually, but preferred, theretaining material for the freeze dried chlorine compounds should betypically pre-treated, such as by soaking the material, like the spongeor other collagen matrices, in a solution of stabilizedchlorine-containing components in buffers with a pH of about 7.5 to 9.0to prevent decomposition of the active substance.

The production of strong, oxidizing environment represented bychlorine-containing compounds occurs by interaction of substances, suchas sodium chlorite, sodium chlorate, chlorous acid anion, with acid,such as citric acid, in a an aqueous medium. In order to prevent contactof these components within the wound dressing matrix and possible earlydecomposition of chlorine-containing compounds with time, a two layersystem of wound dressing is sued. The upper layer, i.e. the layer awayfrom the wound surface, contains the acid moiety while the layeradjacent to the wound contains the dry chlorine compounds. Both layersconsist of a dressing material, such as collagen sponge, approximately0.4 mm to 1.5 mm thick, or collagen membrane, 0.3 mm to 0.5 mm thick, orany other preformed wound dressing material in a completely dry state.The dressing material is soaked with the appropriate chemical solution,quick frozen in a freezer at -70° C. and placed into a freeze dryer(lyophilizer) to evaporate the fluid. By this method, dry layers containeither active substances or the activating moiety, such as citric acid,at appropriate, effective concentrations.

The two layers are secured together for example, glued together, with abiocompatible adhesive, such as an acrylic adhesive. In order to limitthe drying of the wound and to retain wound fluid within the wounddressing, the upper layer of the dressing is laminated or covered withany commercially available adhesive film that limits vapor permeability.Lamination also facilitates easy attachment of the dressing to cover thewound and intact skin. Examples of such a laminating film or laminatedself-adhesive, non-woven fabric are all polyurethane adhesive filmsavailable commercially. The requirement for such a laminating layer arebiologic, non-toxic elements, hypoallergenic physical vapor permeation0.5 mg-2.0 mg H₂ O/cm² /hr and elasticity.

The lower layer of the dressing impregnated with the active ingredientchlorine-containing compound is activated in situ in the presence of anacid from the adjacent layer. On acidification of the active ingredient,stronger oxidizing substances are formed which exert a very effectivecidal effect. Also, importantly, the rate of formation of the oxidizingsubstances from the active ingredient, i.e. the gaseous chlorine dioxidefrom the lyophilized chlorine compound, is pH dependent and may takehours at pH 6-7 and only seconds at 2-2.5. Sufficient weak, acid-likecitric acid is used in the top layer to change the pH of the lower layerfrom the stabilized pH of 8.5-9.0 to 6 pH or less, e.g. 5-6 pH. Thechlorine-containing compound in the lower layer is made stable withalkali buffer of low buffering capacity pH 8.5-9.0, like sodiumbicarbonate and sodium borate, so that the pH may be easily changed by aweak acid to below 6.0 pH.

Aqueous solutions known as chlorine dioxide solution are commerciallyavailable; however, such solutions typically comprise a mixture ofchlorate, chlorite and hydrochlorous acid alkali stabilized at a pH of8.5-9.0, such as the sodium salts of such compounds. Such solutionsgenerally are prepared by the reaction of chlorous acid and sodiumbicarbonate. Such chlorine-containing, aqueous solutions comprisemixtures of 0.1% to 5.0% by weight of the solution with the mixtures,about 1% to 3% by weight of sodium chlorite, 9% or more of sodiumchlorite and about 6% to 8% chlorous acid at a stabilized pH.

The quick freezing and additional freeze drying of the stabilizedchlorine compounds containing solution is essential, as this moiety istemperature and light sensitive. It is also important to adjust the pHof the dressing material to 8.5-9.5 at which temperature there is nodestabilization and decomposition of the active substance. Finally, thefinal product must be packaged in light and water vapor impermeable bagsto obtain good shelf life of the product. Sterilization of the wounddressing so prepared is usually by gamma radiation, 1.5 to 2.5 Merads.

The invention will be described for the purposes of illustration only inconnection with certain embodiments; however, it is recognized thatvarious changes, modifications, additions and improvement may be made tothe illustrated embodiments and examples, all without departing from thespirit and scope of the invention.

BRIEF DESCRIPTION OF THE DRAWING

The drawing is an illustrated, schematic, sectional view of a wounddressing of the invention applied to a wound.

DESCRIPTION OF THE EMBODIMENTS

The wound dressing 10 of the invention is composed of a firstmoisture-impermeable layer 12, a second collagen, citric acid activatorimpregnated layer 14, a third biocompatible, acrylic, moisture permeableadhesive layer 16, and a fourth collagen, chlorine compound, dry,impregnated layer 18, all layers laminated together and the wounddressing shown covering a wound 20.

EXAMPLE 1

Collagen sponge made by the method presented in U.S. Pat. No. 4,193,813from Mar. 18, 1980 was used. The sponge was crosslinked and wastherefore resilient when wet. The sponge existed as a sheet 0.8 mmthick. The original 3.0 pH of the sponge was changed to pH 8.0 byincubation in 0.5M phosphate buffer. The excess buffer was removed bymechanical force. Such a moist sponge binds 35 ml of fluid per 1 g ofthe sponge substance.

The sponge layers were soaked in a 1% solution of chlorine dioxide(DURA-KLOR, a trademark of RioLinda Chemical Co., Inc. of Sacramento,Calif.). When completely hydrated, they were individually frozen at -70°C. in Revco deep freezer for two hours. Frozen layers were then placedin a Virtis freeze dryer, and under vacuum at low temperature, the fluidwas removed from the sponge matrix.

The lyophilizer conditions were: original vacuum between 10 to 50millitorrs, condenser temperature -60° C., sheet was heated to 48° C.Completely dried sponges were kept in light impermeable plastic bags,refrigerated in dry atmosphere. Another set of sponges was soaked assuch (without changing the pH) in 5 weight percent of citric acidsolution. The same procedure was used to dry the sponges.

Two dry sponges, one containing chlorine-active compounds and the othercontaining citric acid were now glued together with adhesive Polytac 212(H & N Chemical Company, New Jersey) by applying a thin layer of theadhesive to one side of either sponge layer. The adhesive fluidevaporated under light compression of the sponges. In the next step, anadhesive film, Tegaderm, was attached to the citric acid-containingsponge surface in such a way that the film made a 1.5 cm wide rimoutside the area of the sponge.

The dry two-layer sponge dressing was placed and heat sealed into bagsand sterilized with 2.5 Merads gamma radiation.

EXAMPLE 2

Example 2 shows the antimicrobial effectiveness of the Example 1dressing in vitro system using E coli grown on agar plate. The standardprocedure sued by microbiological labs was used. In principle, 2 cmdiameter disks were cut off the dressing of Example 1. One disk wasplaced on agar which has been seeded with E coli and placed in 12 cmwide sterile Petri dishes. After a defined incubation time, a clear areaof no growth under or around the disk was determined as an indication ofantibacterial activity of the chlorine-containing moiety in collagensponge system. Once the disk of the final dressing is laced onto the E.coli inoculated agar, 2 ml of a culture media consisting of:

1. Soybean Case Digest Broth (SCDB)

2. Sabouraud's Dextrose Broth (SDB)

3. Soybean Case Digest Agar (SCDA)

4. Soybean Case Digest Agar with 5% sheep blood

5. Sabouraud's Dextrose Agar (SDA)

6. Sabouraud's Dextrose Agar with 5% sheet blood

is added over the disk. These plates are then refrigerated overnight at4° C.-5° C. to prevent diffusion of the chemical into the agar, prior tothe incubation at 37° C. for 18-25 hours, permitting the bacterialgrowth. The presence of the diffused chemicals in the agar inhibits orcompletely blocks the bacterial growth is referred to as an inhibitionzone.

The results of the test performed under the above conditions, with 24hours incubation, showed a 10 cm to 11 cm wide inhibition zone. Thus,the bacteria grew only at the rim of the Petri dish. It was concludedthat the antimicrobial dressing was most effective against the growth ofE. coli.

EXAMPLE 3

Example 3 documents the changes in the antimicrobial activity of thewound dressing with time.

The same testing protocol as shown in Example 2 was used. In this study,the disks were incubated first in plain Petri dishes containing 10 μl ofsterile saline placed on a slowly tilting platform (10 cycles/min). Thedisks were incubated for 0, 6, 12 and 24 hours, removed at three timesfrom the dish and placed on agar inoculated with Staphylococcus aureus.The same procedure was used, viz., 24 hours incubation at 4° C. to allowdiffusion of the active antimicrobial substance was followed by another24 hours incubation at 37° C. All samples at each time period weretested in triplicates.

The results shown in Table I document that the collagen sponge wounddressing with the system described in Example 1 was effective even after24 hours. The variability of the results (at 6 hours) is explained bysample variability.

                  TABLE I                                                         ______________________________________                                        THE DURATION OF ANTIMICROBIAL EFFECTIVENESS                                   OF THE MEDICATED COLLAGEN SPONGE DRESSING                                     AGAINST ST. AUREUS GROWTH                                                     Time    Zone                                                                  (hrs)   Inhibition (cm)                                                       ______________________________________                                        0       10                                                                    6       7.5                                                                   12      9                                                                     24      10                                                                    ______________________________________                                    

EXAMPLE 4

Example 4 documents the determination of the optimal dose ofchlorine-containing moiety in the collagen sponge effectively killingbacteria without an adverse effect on the wound healing.

Collagen sponge dressing was prepared as shown in Example 1 except thatthe strength of original stabilized chlorine compounds containingsolution soaking individual collagen sponges ranged from 0.2, 0.5, 1.0and 2.5 weight percent. After attaching sponges with antimicrobialagents together with sponges containing the activator, the same 2 cmwide disks were dissected and tested for antimicrobial activity by amethod outlined in Experiments 2 and 3. Staphylococcus aureus was usedin agar.

The same collagen sponges with four different dosages of chlorinesubstances were administered on standardized split thickness woundsinflicted in male Yorkshire pigs, 40 lb. weight. The method used wasthat of Chvapil et al (1987). In principle, in anesthetized pigs theskin was cleaned with Betadine and prepped with ethanol. Using anelectrokeratome, a total of 20 shallow wounds, 2.2×2.2×0.4 cm wereexcised on the back of the pig. The wounds were treated with varioustypes of collagen sponges, without (control) or with different doses ofchlorine dioxide. After administration of the dressing onto the wound,the sponge was moistened with 0.5 ml sterile saline, and covered withadhesive film (Op-site, Smith and Nephew). After 56 hours, the dressingwere removed, the wounds excised and processed for histology, 8 randomsections were made of each wound and hematoxylin-eosin stained sectionswere evaluated for the presence of the epithelial cover on the woundsurface. The percent of epithelial cover from the total length of thewound was evaluated. If the results are expressed in percent change ofthe epithelialization as compared to control wounds, it becomes clearthat only the highest dose of chlorine dioxide treated spongesstatistically significantly inhibited the wound healing.

Dressings containing 1% chlorine-containing compounds had no or minimaladverse effect on epithelial cell growth, still at this dose a strikingantimicrobial activity was seen by the inhibition zone (see Table II).During the various steps of manufacturing the final wound dressing, thecontent of the chlorine compounds was reduced by approximately 50%. Itwas found that this loss is attributed mainly to freeze drying invacuum. The actual content of the major chlorine-containing compound(NaClO₂) in the final lyophilized sponge was found to be 100-200μmoles/inch² of the dressing.

                  TABLE II                                                        ______________________________________                                        EFFECT OF VARIOUS DOSE OF CHLORINE-ACTIVE                                     COMPONENTS IN COLLAGEN SPONGE WOUND DRES-                                     SING ON THE BACTERIAL GROWTH AND RATE OF                                      EPITHELIALIZATION OF SHALLOW WOUNDS                                           IN PIG MODEL                                                                              Antimicrobial                                                     Concentrate Inhibition Zone.sup.2                                                                      Effect on Healing.sup.1                              %           (cm)         (% Inhibition)                                       ______________________________________                                        0           0                0                                                0.2         2-5              0                                                0.5         7-10      x      0                                                1           10-11     x      0-5                                              2.5         11-12     x      25-30    x                                       ______________________________________                                         x Refers to statistical significant inhibition at p < 0.01.                   .sup.1 Data presented in % of change from control values and are average      of 3 wounds analysis for each treatment modality                              .sup.2 The lowest and highest value (range) of inhibition zone is shown. 

What is claimed is:
 1. An antimicrobial wound dressing, which wounddressing comprises:a) a first layer of a retaining dressing materialselected from the group consisting of: open cell foam materials,membrane materials, woven materials and non-woven materials impregnatedwith an antimicrobial effective amount of stabilized, dry, lyophilizedchlorine-containing compounds which on activation generate achlorine-oxidizing compound, one side of the first layer to be placed incontact with a wound to be treated; and b) a second layer of a retainingdressing material adjacent to the first layer, said second layerimpregnated with an amount of dry organic acid compound effective toactivate said chlorine-containing compounds wherebywhen the first layerof the wound dressing is placed on the wound being treated the presenceof moisture from the wound causes, the dry organic acid compound toactivate the dry chlorine-containing compounds to produce an antiviral,antimicrobial chlorine oxidant to treat the wound.
 2. The dressing ofclaim 1 which includes a third layer of a vapor-impermeable materialwhich is adjacent to said second retaining dressing layer, wherein thethird layer retains wound moisture within the wound dressing, said thirdlayer having a physical vapor permeation of from 0.5 mg to 2.0 mg H₂O/cm² hr.
 3. The dressing of claim 1 which includes a layer ofmoisture-permeable adhesive material to secure the adjacent surfaces ofthe first and second layer together.
 4. The dressing of claim 1 whereinthe retaining material comprises a collagen material.
 5. The dressing ofclaim 1 wherein the acidic compound is selected from a group consistingof: ascorbic acid; tartaric acid; boric acid; and citric acid.
 6. Thedressing of claim 1 wherein the retaining dressing material ispre-treated with a solution having a pH of from about 8.0 to 9.0 toprevent decomposition of the stabilized, dry, lyophilized,chlorine-containing compounds on impregnation.
 7. The dressing of claim1 wherein the chlorine-containing compounds comprise a mixture of sodiumchlorite and sodium chlorate.
 8. The wound dressing of claim 1 which hasbeen sterilized.
 9. The dressing of claim 1 wherein each layer of theretaining dressing material has a thickness ranging from about 0.4 mm to1.5 mm, and the retaining dressing material has been impregnated withfrom about 0.1 to 5.0 weight percent of a chlorine dioxide solution. 10.The dressing of claim 1 wherein the second retaining dressing layer hasbeen prepared employing an aqueous solution of from about 2% to 7% byweight of a weak, organic acid compound.
 11. An antimicrobial wounddressing prepared by the steps comprising:a) impregnating a firstretaining dressing material selected from the group consisting of: opencell foam materials, membrane materials, woven materials and non-wovenmaterials, with an alkali-stabilized aqueous chlorine dioxide solutionwhich on activation generates a chlorine dioxide gas; b) impregnating asecond retaining dressing material with an aqueous solution of an acidiccompound capable of forming a dry acidic compound in an effective amountto act as an activator for the chlorine dioxide; c) freeze drying thefirst and second dressing material to obtain a first retaining dressingmaterial impregnated with dry stabilized chlorine dioxide and a secondretaining dressing material impregnated with a dry acidic compound; andd) placing the adjoining surface layers of the first and secondretaining dressing materials together with the opposite surface of thefirst retaining dressing material to be placed against the wound to betreated, whereby in the presence of moisture from the wound the acidiccompound activates the dry chlorine dioxide to produce in situ, anantimicrobial, gaseous, chlorine dioxide to treat the wound.
 12. Thewound dressing of claim 11 wherein the dry chlorine dioxide ranges fromabout 0.5 to 1.5 percent by weight of the first layer material.
 13. Thewound dressing of claim 11 which includes impregnating the retainingdressing material of the first layer material with an alkali-stabilizedsolution of sodium chlorate, sodium chlorite and chlorous acid andfreeze drying the impregnated dressing material.
 14. The wound dressingof claim 11 wherein the first and second layer material comprises acollagen material.
 15. The wound dressing of claim 11 wherein the dryacidic acid compound is selected from the group consisting of: ascorbicacid; tartaric acid; boric acid; and citric acid.